FIGURE 3.

Absorption (A), CD (B), and EPR (C) spectra of rCueO (black) and mutants, C500S (blue), C500S/D112N (red), E506Q (purple), and C500S/E506Q (green). Absorption and CD spectra of ∼100 μm proteins in 0.1 m phosphate buffer, pH 7, were measured at room temperature using a 1-cm path length quartz cell. The units of the ordinate are based on the protein molecule. EPR spectra were measured at 77 K, with a frequency of 9.2 GHz, microwave power of 4 milliwatts, modulation of 1 mT at 100 kHz, filter of 0.3 s, sweep time of 4 or 8 min, and amplitude of 200–400. The EPR spectra are normalized to give analogous signal intensities except for C500S/E506Q. The total numbers of the EPR-detectable Cu²⁺ in rCueO, C500S, C500S/D112N, E506Q, and C500S/E506Q are 2.0, 1.0, 1.0, 1.0, and 0.36/protein molecule, respectively.